The detection and quantification of chemical and biochemical components in aqueous fluids, in particular biological fluids, such as, whole blood and urine and biological fluid derivatives, such as, blood serum and blood plasma, is of ever-increasing importance. Important applications exist in medial diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals and the like. In some instances, the amount of materials being determined are either so small or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case, the results are generally not available for hours or days after sampling. These apparati are not for suitable for operation by patients who often need to perform such tests routinely, quickly and reproducibly outside of a laboratory setting with rapid or immediate information display.
Devices and methods are generally known for detecting analytes, such as glucose, in a blood sample. For example, one common medical test is the measurement of blood glucose levels by diabetics. Current diabetes maintenance programs encourage patients to measure their blood glucose level from two to seven times a day, depending on the nature and severity of the disease. Based on the observed pattern in the measured glucose levels, the patient and physician make adjustments in diet, exercise and insulin intake to better manage the disease. Clearly, the accuracy and immediacy of the results of such tests is paramount.
Thus, the art is replete with examples of methods for detecting glucose in a blood sample. For example, U.S. Pat. No. 5,179,005 to Phillips et al. discloses a method for determining the presence of an analyte in a fluid. In this method, a MBTH-DMAB (3-methyl-2-benzothiazolinone hydrazone hydrochloride and 3-dimethylaminobenzoic acid) dye forms a chromophore when reacted with an analyte such as, for example, glucose. This dye absorbs at approximately 635 nm but does not absorb to any significant extent at 700 nm. Because blood contains background materials which absorb at both 635 nm and 700 nm, this method requires that a relationship between absorbants at 635 nm and 700 nm due to blood contaminants be determined by measuring the absorbance of blood samples with 0 mg/dl glucose over a wide range of blood colors. Such a color range was constructed by varying the hematocrit of the blood which results in substantially linear relationships. From these relationships, contaminating absorbance readings at 700 nm are normalized to the equivalent contaminating absorbant readings at 635 nm. This normalization was expressed as K/S-15n=(K/S-15.multidot.1.54)-0.133. Using this relationship, as well as others, contaminating absorbencies at 635 nm were accounted for and subtracted out to give a true glucose concentration. Such a method is limited, however, because the selected dye must absorb at a wavelength that is different from the wavelength at which red blood cells absorb. Furthermore, the need to construct a standard for the background materials in blood is time consuming and expensive.
A similar test for glucose is disclosed by U.S. Pat. No. 5,426,032 to Phillips et al. which describes a no-wipe whole blood glucose test strip. This test strip is adapted for use in a reflectance reading apparatus that is capable of measuring reflectance at two different wavelengths. This patent is similar to Phillips' earlier issued '005 patent because a background absorbance reading must be taken at 700 nm while the reflectance of the chromophore is read at 635 nm. Thus, in the '032 patent when measuring analytes contained in whole blood, readings are taken at two different wavelengths. The reading at one wavelength, e.g. 700 nm, is used to subtract out the background interference caused by, for example, hematocrit, blood oxygenation, and other variables which may affect the result. The method of this patent, however, is limited in that the signal-producing reagent must be absorbed at a wavelength other than a wavelength at which the assay medium substantially absorbs. Furthermore, this method is unable to correct for contaminants and/or other analytes which absorb at the chromophore's wavelength, i.e., 635 nm, and at the same time the chromophore produced by the analyte also absorbs at a second wavelength, i.e., 700 nm.
In a slightly different approach to correcting for contaminating signals during analyte detection in a sample, U.S. Pat. No. 5,453,360 to Yu describes an oxidative coupling dye for photometric quantitative analysis of such analytes. In particular, a dye couple is described which includes 3-methyl-2-benzothiazolinone hydrazone (NBTH) and 8-aniline-1-naphthalenesulfonate (ANS). This dye couple is used as an indicator in a reaction cascade that produces a strong oxidizing agent, such as, hydrogen peroxide. This strong oxidizing agent then reacts with the dye couple to produce a blue dye reaction product. The NBTH-ANS dye couple exhibits strong and flat spectral absorption at about 600 to 650 nm. This region of absorbance is free of blood color interference. Accordingly, glucose and other analytes that react with an oxidase enzyme to produce the above-described strong oxidizing agent can be accurately measured without much optic calibration. This patent is limited because the dye couple must absorb in an area of the reflectance spectrum between 600 and 650 nm which is outside the region of blood color interference or the dye couple must be used in the absence of red blood cells so that it would not to be subject to interference by the color of the blood. More generally, this patent suffers from the drawback that unknown components in a sample which happen to absorb at the detection wavelength will cause the analyte measurements to be over-estimated.
Similarly, U.S. Pat. No. 5,389,524 to Larsen et al. describes a method and an apparatus for quantitatively monitoring a chemical component dissolved in a liquid medium. This patent describes taking two measurements of a system at the same wavelength. The absorbance of a colored reaction product is determined by subtracting an end-point absorbance from a background absorbance. Thus, this patent takes two absorbance measurements at the same wavelength and is susceptible to erroneous results if one or more analytes or interfering substances happen to absorb at the same wavelength that the two absorbance measurements are taken.
Non-glucose analyte detection systems are also well known in the art. For example, U.S. Pat. No. 5,204,242 to Junius-Comer describes the use of a substituted phenol with high stability and low non-specific reactivity as a coupling component used in a colormetric process for detecting oxidative coupling reactions, such as the determination of enzymatic creatinine levels. In this method, a reference measurement is made at 700 nm before the reaction starts; while the extinction increase is measured at 546 nm between the sixth and tenth minute of reaction. This method is limited by its assumption that no interfering component absorbs at the detection wavelength, i.e., 546 nm.
It is also known in the art to use fluorescent or other detection schemes for determining the presence and/or quantity of an analyte in a sample. For example, U.S. Pat. No. 5,527,684 to Mabile et al. describes a method of measuring the luminescence emitted in a luminescent assay. This method includes forming a reaction mixture by contacting a sample with an internal reference compound that emits at a first wavelength and a tracer compound that emits at a second wavelength. The sample is then irradiated at a single excitation wavelength. The tracer is detected by measuring the emitted luminescence at the second wavelength. The internal reference is detected by measuring the emitted luminescence at the first wavelength. Using the luminescence emitted at the first wavelength by the internal reference, a correction is made to the luminescence emitted at the second wavelength by the tracer. In this way, the analyte in question can be quantified or detected.
In summary, the prior art methods cited above all suffer from the drawback that they are susceptible to over-estimating and/or providing false positive results if a contaminating component of a sample happens to absorb at the detection wavelength. Thus, there is a need for an improved analyte detection system which is able to compensate for interfering absorbencies and to provide accurate detection and/or quantification of analytes in a sample. In particular, there is a need for quick, cost-effective, accurate and easy-to-use methods and systems for indirectly determining the presence and/or quantity of one or more analytes in a system. The present invention is directed to meeting these and other needs.